Melatonin Protects against Lung Fibrosis by Regulating the Hippo/YAP Pathway

Xiaoguang Zhao, Jian Sun, Wei Su, Huitong Shan, Bowen Zhang, Yining Wang, Azaliia Shabanova, Hongli Shan, Haihai Liang, Xiaoguang Zhao, Jian Sun, Wei Su, Huitong Shan, Bowen Zhang, Yining Wang, Azaliia Shabanova, Hongli Shan, Haihai Liang

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive, fibrotic interstitial pneumonia with high mortality. Melatonin, a hormone predominantly secreted by the pineal gland, has been reported to participate in the process of IPF. However, the mechanisms underlying the effect of melatonin in pulmonary fibrosis have not been elucidated to date. This study was designed to evaluate the anti-fibrotic role of melatonin in pulmonary fibrosis and to elucidate the potential mechanisms. We observed that melatonin markedly attenuated bleomycin (BLM)-induced experimental lung fibrosis in mice and inhibited TGF-β1-induced fibrogenesis in lung fibroblasts. Additionally, we determined that luzindole, a melatonin receptor inhibitor, reduced the anti-fibrotic effect of melatonin. Further studies showed that melatonin alleviated the translocation of YAP1 from cytoplasm to nucleus, a key downstream effector of the Hippo pathway, in vivo and in vitro by interacting with its receptor. Taken together, our results suggest that melatonin prevents lung fibrosis by inhibiting YAP1 and indicate that melatonin replacement could be a novel strategy for the treatment of lung fibrosis.

Keywords: YAP1; idiopathic pulmonary fibrosis; melatonin.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Melatonin alleviates bleomycin-induced pulmonary fibrosis in mice. (A) Representative microscopy images of Masson’s staining of lung tissue derived from BLM-treated mice or BLM + Melatonin mice; scale bar, 50 μm; (B) Quantification of total interstitial fibrotic area using Image-Pro Plus; (C) IHC staining shows the differential expression of fibronectin 1 (Fn1). n = 6 mice in each group, red arrow indicated the expression of Fn1. The mRNA expression of Col 1α1 (D) and Col 3α1 (E) was measured via qRT-PCR. β-Actin mRNA acted as the internal control; (F) Collagen content of lung tissues was detected by SircolTM Soluble Collagen Assay; (G) Western blot analysis examining the fibrotic-related protein expression in BLM-treated mice with or without melatonin injection; (H) The statistical Western blotting data. n = 4 mice in each group. Values are the mean ± SEM of five independent experiments. * p < 0.05.
Figure 2
Figure 2
Identification of melatonin receptor as a functional target in melatonin-mediated fibrosis. mRNA expression of Col 1α1 (A) and Col 3α1 (B) was obtained with qRT-PCR. β-Actin was used as the internal control. (C,D) Wound healing assay demonstrated that luzindole abolished the TGF-β1-induced cell migration and the Western blot (E,F) analysis of fibrosis-related proteins illustrated that luzindole blocked the inhibitory effect of melatonin in TGF-β1-induced fibrogenesis. β-Actin served as the loading control. Values are the mean ± SEM of five independent experiments. ns: not significant; * p < 0.05.
Figure 3
Figure 3
Luzindole suppresses the effect of melatonin in lung fibrogenesis along with the change of cell proliferation and myofibroblasts activation. (A) The analysis of EDU staining demonstrated the cell proliferation ability of lung fibroblasts with different treatments; (B) The statistical diagram of the EDU assay; (C) Immunostaining of α-SMA in lung fibroblasts demonstrated the inhibitory effect of luzindole. Values are the mean ± SEM of five independent experiments. * p < 0.05.
Figure 4
Figure 4
Dysfunction of YAP1 is involved in the anti-fibrotic effect of melatonin in BLM-treated mice. (A) The expression of YAP1 was down-regulated after treatment with melatonin, determined with immunohistochemical staining; red arrow indicated the expression of YAP1. (B) qRT-PCR analysis of the mRNA expression of YAP1 in BLM-mice. β-Actin mRNA acted as the internal control; n = 6 mice in each group. (C,D) Western blot analysis of YAP1 and the related statistical data are shown. (E) Immunofluorescence assay to determine the effect of melatonin and luzindole on the location of YAP1 in lung fibroblasts. qRT-PCR (F) and Western blot (G,H) assays were performed to evaluate the effect of melatonin and luzindole on the mRNA and protein level of YAP1. β-Actin served as the internal control. Values are the mean ± SEM of five independent experiments. * p < 0.05.
Figure 5
Figure 5
Forced expression of YAP1 abrogated the inhibitory effect of melatonin on production of collagen. (A,B) Real-time RT-PCR analysis showing that YAP1 overexpression blunted the inhibitory effects of melatonin on TGF-β1-induced up-regulation of Col 1α1 and Col 3α1 at the mRNA level. n = 6 cell batches. Representative photomicrographs showing that YAP1 restored the cell migration ability (C,D) compared with the TGF-β1+melatonin group. (E,F) Western blotting was used to determine the effect of YAP1 overexpression on the inhibitory effects of melatonin in TGF-β1-induced up-regulation of pro-fibrotic proteins, including collagen 1, Fn1, α-SMA and Twist1.Values are the mean ± SEM of five independent experiments. * p < 0.05.
Figure 6
Figure 6
Overexpression of YAP1 reversed the suppression of cell proliferation and myofibroblasts activation. Microphotograph of EDU staining (A,B) and α-SMA positive staining (C) illustrated forced expression of YAP1 could rescue the anti-fibrotic effect of melatonin. (D) The schematic diagram of this study, melatonin alleviated lung fibrosis in vivo and in vitro via interacting with its receptor, and this anti-fibrotic effect was mediated by YAP1 inactivation. Values are the mean ± SEM of five independent experiments. * p < 0.05.

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